1. Field of the Invention
The present invention relates to a system and method for separating particles. The invention has particular advantages in connection with separating blood components, such as antigen-specific blood cells.
This application is related to U.S. Pat. No. 5,674,173, issued on Oct. 7, 1997; U.S. patent application Ser. No. 08/423,583, filed on Apr. 18, 1995 (abandoned); U.S. patent application Ser. No. 08/634,167, filed on Apr. 18, 1996 (pending); and U.S. patent application Ser. No. 09/009,378, filed on Jan. 20, 1998 (pending). The entire disclosure of this U.S. patent and the entire disclosures of these U.S. patent applications are incorporated herein by reference.
2. Description of the Related Art
In many different fields, liquids carrying particle substances must be filtered or processed to obtain either a purified liquid or purified particle end product. In its broadest sense, a filter is any device capable of removing or separating particles from a substance. Thus, the term "filter" as used herein is not limited to a porous media material but includes many different types of processes where particles are either separated from one another or from liquid.
In the medical field, it is often necessary to filter blood. Whole blood consists of various liquid components and particle components. Sometimes, the particle components are referred to as "formed elements." The liquid portion of blood is largely made up of plasma, and the particle components primarily include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). While these constituents have similar densities, their average density relationship, in order of decreasing density, is as follows: red blood cells, white blood cells, platelets, and plasma. In addition, the particle constituents are related according to size, in order of decreasing size, as follows: white blood cells, red blood cells, and platelets.
Although blood is primarily made up of white blood cells, red blood cells, and platelets, there are a number of other particle components of blood. For example, blood includes T-cells (a type of white blood cell), B-cells, monocytes (another type of white blood cell), stem cells, and NK cells. Most of these particles have similar sizes and/or densities. However, some of these particles have different surface chemistry characteristics designated with a specific "CD+" marker or symbol. For example, T-cells include CD2+ cells, CD3+ cells, CD4+ cells, and CD8+ cells; B-cells include CD9+ cells, CD10+ cells, and CD19+ cells; monocytes include CD14+ cells; stem cells include CD34+ cells; leukocytes include CD45+ cells; and NK cells include CD56+ cells. Corresponding antibodies, identified with an "anti-CD" marker or symbol, are capable of binding to these cells having particular surface chemistry characteristics. For example, anti-CD2 is capable of binding with CD+2 cells; anti-CD3 is capable of binding with CD3+ cells; and anti-CD4 is capable of binding with CD+4 cells. In a similar manner, anti-CD8, anti-CD9, anti-CD10, anti-CD14, anti-CD15, anti-CD19, anti-CD20, anti-CD34, anti-CD38, anti-CMRF44, anti-CD45, anti-CD56, anti-CD83, anti-glyocophorin, anti-cytokeratin, and anti-EPCAM are capable of binding with CD8+ cells, CD9+ cells, CD10+ cells, CD14+ cells, CD15+ cells, CD19+ cells, CD20+ cells, CD34+ cells, CD38+ cells, CMRF44+ cells, CD45+ cells, CD56+ cells, CD83+ cells, glyocophorin+ cells, cytoketatin+ cells, and EPCAM+ cells, respectively, for example.
Most current separation devices rely on density and size differences or surface chemistry characteristics to separate and/or filter blood components for transfusion or reinfusion purposes. Typically, blood components are separated or harvested from other blood components using a centrifuge. The centrifuge rotates a blood reservoir to separate components within the reservoir using centrifugal force. In use, blood enters the reservoir while it is rotating at a very rapid speed and centrifugal force stratifies the blood components, so that particular components may be separately removed. Although some centrifugal separation techniques are effective at separating some blood components from one another, many centrifugal separation processes are not capable of producing a highly purified end product.
In one type of separation procedure, a peripheral blood collection (withdrawn from an artery or vein) or a bone marrow blood collection is purified in a centrifugal separation process to isolate what is known as a peripheral blood cell collection or bone marrow blood cell collection, respectively. The peripheral blood cell collection or bone marrow blood cell collection primarily includes plasma, red blood cells, white blood cells (leukocytes, such as T-cells and monocytes), and stem cells. These cell collections also may include amounts of B-cells and NK cells.
After undergoing a therapeutic treatment, such as chemotherapy or radiation therapy, to eliminate cancerous tumor cells, cancer patients often receive a transfusion of a peripheral blood cell collection or a bone marrow blood cell collection to replace stem cells destroyed as a side effect of the treatment. To reduce risks associated with infusing blood components from a foreign donor, some of these transfusions are autologous, where blood components collected from the patient are later reinfused back to the patient. Another type of transfusion, known as allogenic transfusion, involves collecting blood components from a donor and then infusing the collected blood components into a recipient who is different from the donor. Sometimes, however, the recipient of an allogenic transfusion experiences a disease commonly known as graft versus host disease. In graft versus host disease, particular T-cells, which may accompany the blood components, are infused into the recipient and "recognize" that the recipient's body is "foreign" from that of the donors. These T-cells "attack" healthy cells in the recipient's body, rather than performing a normal immunological protective function. Recent studies have shown that a particular type of T-cells, namely CD8+ cells, could cause graft versus host disease.
Several prior attempts to separate stem cells from T-cells prior to reinfusion have been made. In one separation method, a selective antibody, anti-CD34, is introduced into a collected blood component sample after separating a substantial number of platelets and red blood cells from the sample. This selective antibody chemically attaches to stem cells (CD34+ cells) to "mark" them. To separate the marked stem cells from the remaining blood components, the collected blood components are passed between stationary beads coated with a material which chemically bonds with the selective antibody. These chemical bonds retain the marked stem cells on the beads to filter them from the remaining blood components.
To remove the marked stem cells from the beads, an operator agitates the beads or flushes a chemical solution through the beads to break the chemical bonds between the material and selective antibody. This separation process, however, is extremely expensive, tedious, and time consuming and often requires an initial centrifugation procedure to remove platelets and red blood cells. In addition, sometimes the beads do not remove a significant number of stem cells, and a substantial number of T-cells often remain in the separated end product.
In another type of separation procedure, magnetic particles or fluid having an attached antibody, anti-CD34, are added to a blood component collection. The antibody binds with stem cells (CD34+ cells) in the sample to link the magnetic particles and stem cells together. To separate the stem cells, a magnetic separator is used to attract the magnetic substance and stem cells, and linked a substance is then added to break the chemical bonds between the stem cells and magnetic substance.
Although this separation procedure is capable of separating some stem cells and T-cells, it is expensive and labor intensive. A significant number of T-cells remain in the separated end product and a sizable number of stem cells are not recovered. In addition, the substances added to the blood sample in both the bead separation process and the magnetic separation process are potentially toxic if they are infused along with the separated blood components.
Centrifugal elutriation is another process used to separate T-cells from stem cells. Normally, this process is used to separate cells suspended in a liquid medium without the use of chemical antibodies. In one common form of elutriation, a cell batch is introduced into a flow of liquid elutriation buffer. This liquid which carries the cell batch in suspension, is then introduced into a funnel-shaped chamber located on a spinning centrifuge. As additional liquid buffer solution flows through the chamber, the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber having the greatest centrifugal force.
When the centrifugal force and force generated by the fluid flow are balanced, the fluid flow is increased to force slower-sedimenting cells from an exit port in the chamber, while faster-sedimenting cells are retained in the chamber. If fluid flow through the chamber is increased, progressively larger, faster-sedimenting cells may be removed from the chamber.
Thus, centrifugal elutriation separates particles having different sedimentation velocities. Stoke's law describes sedimentation velocity (SV) of a spherical particle, as follows: ##EQU1##
where, r is the radius of the particle, .rho..sub.p is the density of the particle, .rho..sub.m is the density of the liquid medium, .eta. is the viscosity of the medium, and g is the gravitational or centrifugal acceleration. Because the radius of a particle is raised to the second power in the Stoke's equation and the density of the particle is not, the size of a cell, rather than its density, greatly influences its sedimentation rate. This explains why larger particles generally remain in a chamber during centrifugal elutriation, while smaller particles are released, if the particles have similar densities.
As described in U.S. Pat. No. 3,825,175 to Sartory, centrifugal elutriation has a number of limitations. In most of these processes, particles must be introduced within a flow of fluid medium in separate discontinuous batches to allow for sufficient particle separation. Thus, some elutriation processes only permit separation in particle batches and require an additional fluid medium to transport particles. In addition, flow forces must be precisely balanced against centrifugal force to allow for proper particle
Further, a Coriolis jetting effect takes place when liquid and particles flow into an elutiation chamber from a high centrifugal field toward a lower centrifugal field. The liquid and particles turbulently collide with an inner wall of the chamber facing the rotational direction of the centrifuge. This phenomenon mixes particles within the chamber and reduces the effectiveness of the separation process. Coriolis jetting also shunts flow of liquid and particles along the inner wall of the elutriation chamber from the inlet directly to the outlet. Thus, particles pass around the elutriative field to contaminate the end product.
If the combined density of particles and liquid in the elutriation chamber is significantly greater than the density of liquid entering the chamber, Coriolis jetting increases. This is because the relatively low density liquid entering the elutriation chamber encounters increased buoyant forces tending to accelerate the flow of liquid toward the outlet of the elutriation chamber. When the accelerated flow of liquid encounters tangential forces in the chamber, the flow of liquid may form a Coriolis jet capable of carrying larger, relatively faster sedimenting particles around the elutriative field and through an outlet of the chamber.
Particle mixing by particle density inversion is an additional problem encountered in some prior elutriation processes. Fluid flowing within the elutriation chamber has a decreasing velocity as it flows in the centripetal direction from an entrance port toward an increased cross-sectional portion of the chamber. Because particles tend to concentrate within a flowing liquid in areas of lower flow velocity, rather than in areas of high flow velocity, the particles concentrate near the increased cross-sectional area of the chamber. Correspondingly, since flow velocity is greatest adjacent the entrance port, the particle concentration is reduced in this area. Density inversion of particles takes place when the centrifugal force urges the particles from the high particle concentration at the portion of increased cross-section toward the entrance port. This particle turnover reduces the effectiveness of particle separation by elutriation.
For these and other reasons, there is a need to improve particle separation.